Thesis

Characterization of an aminopeptidase gene expressed in akinetes, vegetative, hormogonia and heterocysts of Nostoc punctiforme

Nostoc punctiforme is a filamentous cyanobacterium capable of multiple cellular differentiation from vegetative cells into akinetes, heterocysts and hormogonia. In order to control this differentiation, tight regulation of gene expression is required. Previously, a model system was developed for Nostoc punctiforme involving a glucose-6-dehydrogenase mutant strain of N. punctiforme (zwf mutant) that forms akinetes at high frequencies following a switch from photoautotrophic to dark heterotrophic growth condition in the presence of fructose. From this model system, a hormogonia/akinete peptidase (aapN) gene was identified as an up-regulated gene during akinete formation by differential display and microarray analysis. A reporter strain of aapN was created by transcriptional fusion of the promoter region to a promoterless gfp-gene in the pSUN119 plasmid, which further showed transcriptional expression specific to akinetes and hormogonia (Argueta et al., 2006). Random amplification of cDNA ends (RACE) analysis was used to identify the transcriptional start sites for the aapN gene as well as two transcriptional start sites for the upstream putative dnaK heatshock gene (NpR5998). ix Bioinformatic analysis of orthologous genes in closely related cyanobacteria identified conserved sequences in the intergenic region upstream of the +1 transcriptional start site suggesting putative relevancies to transcriptional control. To elucidate the important areas of the promoter region for these for transcriptional or translational control, a series of labeled promoter fragments of increasing length were fused to GFP in the pSUN119 plasmid and electroporated into N. punctiforme wild-type to create a series of reporter strains. Epifluorescence microscopy following akinete induction indicated the cis-acting regulatory element required for silencing gene transcription was located downstream in the aapN open reading frame. Experimental results from the reporter plasmids (GFP fused with aapN promoter fragments) showed that the smaller fragments (59 bp - 381 bp) had zero capacity at silencing gene transcription regardless of cell type (akinete, heterocyst, hormogonia, and vegetative). It was a 516 basepair promoter fragment that reached 240 basepairs past the putative aapN translational start site and into the open reading frame length that achieved tight negative regulation of gene transcription. Subsequent longer promoter fragments repeated the same result of tight negative regulation.

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